Calcineurin is a calcium-dependent, serine/threonine phosphatase that is a signal transduction mediator involved in a variety of pathways identified in T cells. There are three isoforms of the catalytic subunit of calcineurin-α, β, and γ. Unique and distinct roles for the α and β isoforms have been identified. Importantly, the β isoform appears to be the primary isoform required for normal activity of T cells.
The addition of the calcineurin inhibitors cyclosporine A and FK506 to immunosuppressive regimens reduces the incidence of acute allograft rejection and effectively doubles one-year survival of kidney transplant patients. However, long-term graft survival has improved far less significantly, with only 66% and 78% of deceased-donor kidney and living-donor kidney recipients, respectively, surviving 5 years. This statistic is even more striking when considered for different racial groups. Eighty percent of Caucasians who receive living-donor organs survive for 5 years while only 64% of African Americans live that long. Similar trends are observed for recipients of deceased-donor organs; there is a 70% survival rate for Caucasians but only 55% for African Americans. Understanding mechanisms that contribute to disparate outcomes for transplant patients is an area of tremendous importance. Despite considerable effort, no consensus on the underlying causes has been reached that adequately explains racial disparities in long-term outcomes.
Cyclosporine A (cyclosporine A) and FK-506 (tacrolimus) are structurally unrelated compounds that form drug-receptor complexes with immunophilins (cyclophilin-18 and FKBP-12 respectively) and potently inhibit calcineurin phosphatase activity. The wide spread use of cyclosporine A and tacrolimus in the past two decades has markedly reduced the frequency of acute allograft rejection and prolonged patients survival. Despite their proven benefits, monitoring of cyclosporine A and tacrolimus levels in a patient has proven to be a poor clinical indicator of transplant outcomes. Some patients experience rejection in the presence of adequate or even excessive blood concentrations (Caruso et al., (2001) Clin. Chem. 47: 1679-1687), whereas others develop toxicity even when blood trough concentrations are low (Citterio F. (2004) Transplant. Proc. 36: 420S-425S; Kahan B. D. (2004) Transplant. Proc. 36: 378S-391S). However, in the absence of an alternative means of monitoring calcineurin inhibitor efficacy, current treatment protocols continue to rely upon plasma drug levels for therapeutic monitoring and optimizing immunosuppression.
One potential alternative to plasma drug level monitoring is direct assay of calcineurin activity. However, few studies have directly examined calcineurin activity in T cells or investigated the effects of calcineurin inhibitors on enzyme activity. Studies of calcineurin activity in vivo have focused on issues including pharmacodynamics in response to cyclosporine and tacrolimus (Koefoed-Nielsen & Jorgensen (2002) Transplant. Proc. 34: 1743-1744; Koefoed-Nielsen et al., (2006) Transpl. Int. 19: 821-827; Koefoed-Nielsen et al., (2005) Transplant. Proc. 37: 1736-1738; Mortensen et al., (2006) Transplant. Proc. 38: 2651-2653.) and possible effects of variables including gender and time of day (Koefoed-Nielsen et al., (2005a) Scand. J. Immunol. 62: 309-311).
In an early study using transplant patients, Batiuk et al., ((1997) J. Clin. Invest. 100: 1894-1901) used a 32PO4-labeled calcineurin specific substrate to measure the effects of cyclosporine A on calcineurin activity in 30 renal allograft recipients. In vivo measurements demonstrated that calcineurin activity was inhibited by up to 80% one hour after an oral dose of cyclosporine A, but only 20-30% within four hours. However, the degree of enzyme inhibition and the effect on cytokine production varied greatly between individuals. In a similar study, Pai et al., ((1994) Blood 84: 3974-3979) examined the long-term effect of cyclosporine A on calcineurin activity in peripheral lymphocytes from bone marrow transplant patients. While cyclosporine A initially inhibited calcineurin activity during the first 100 days of transplantation, enzyme activity progressively rose over time and within 6 months was similar to non-transplant controls.
Calcineurin is unique among phosphatases in that its activity is calcium-dependent and is central to T cell receptor (TCR) signaling and amplification of immune responses. The activation of the TCR complex leads to the release of intracellular calcium and calcineurin-mediated dephosphorylation of transcription factors that regulate IL-2 and other pro-inflammatory cytokines (Macian F. (2005) Nat. Rev. Immunol. 5: 472-484). Calcineurin is known to be activated downstream of the T cell receptor and regulates transcription factors including the Nuclear Factor of Activated T cells (NFATc). NFATc proteins, in turn, control expression of cytokines including IL-2 and IL-4. Blockade of calcineurin/NFAT activity inhibits T cell activity and results in immune suppression. Although cyclosporine A has been clinically used for more than 20 years and FK506 over a decade, appropriate and effective target blood levels for maintenance immunosuppression have yet to be properly defined. Discrepancies between calcineurin inhibitor dose and clinical immune suppression suggest that calcineurin activity itself may be a source of variability. However, there have been only limited studies that directly measure calcineurin activity, and there is no data regarding factors which may affect the calcineurin sensitivity to inhibition by cyclosporine A and FK506.